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Acute and Rapid Degradation of Endogenous Proteins by Trim-Away

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Acute and Rapid Degradation of Endogenous Proteins by Trim-Away Acute and rapid degradation of endogenous proteins by Trim-Away Dean Clift1,2*, Chun So1,3, William A. McEwan2, Leo C. James,2* and Melina Schuh1,2,3* 1These authors contributed equally 2Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom 3Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany *Corresponding authors *Correspondence: [email protected] (D.C), [email protected] (L.J.), [email protected] (M.S.) KEYWORDS Trim-Away; protein degradation; TRIM21; ubiquitin ligase; Fc receptor; antibody; microinjection; electroporation; intracellular immune response; oocyte; mouse oocyte; primary cell; meiosis; EDITORIAL SUMMARY This Protocol describes ‘Trim-away’, an approach for rapid protein depletion in different cell-types. TRIM21-mediated proteasomal degradation is induced by microinjection or electroporation of an antibody against the protein of interest. TWEET A new Protocol describing the detailed procedures for ‘Trim-away’, an approach for rapid protein depletion in oocytes, primary cells, and cultured cells @SchuhLab @mpi_bpc @MRC_LMB @CellBiol_MRCLMB COVER TEASER Rapid protein depletion in cells using ‘Trim-away’ Please indicate up to four primary research articles where the protocol has been used and/or developed. 1. Clift, D. et al. A method for the acute and rapid degradation of endogenous proteins. Cell. 171, 1692-1706 (2017). 1 Abstract Protein depletion is a key approach to understanding functions of a protein in a biological system. We recently developed the Trim-Away approach in order to rapidly degrade endogenous proteins without prior modification. Trim-Away is based on the ubiquitin ligase and Fc receptor TRIM21, which recognizes antibody-bound proteins and targets them for degradation by the proteasome. In a typical Trim-Away experiment, protein degradation is achieved in three steps: first, introduction of an antibody against the target protein; second, recruitment of endogenous or exogenous / overexpressed TRIM21 to the antibody-bound target protein; and third, proteasome-mediated degradation of the target protein, antibody and TRIM21 complex. Protein degradation by Trim-Away is acute and rapid, with half-lives of around 10-20 minutes. The major advantages of Trim-Away over other protein degradation methods are that it can be applied to any endogenous protein without prior modification; that it uses conventional antibodies which are widely available; and that it can be applied to a wide range of cell types including non-dividing primary human cells, where other loss-of- function assays are challenging. In this Protocol, we describe the detailed procedures for antibody preparation and delivery in mouse oocytes and cultured cells via microinjection and electroporation. In addition, we provide recommendations for antibody selection and validation and for generating TRIM21 over-expressing cell lines for cases where endogenous TRIM21 is limited. A typical Trim- Away experiment takes just a few hours. Introduction Protein depletion is one of the key tools for studying protein functions in cells and tissues and can be achieved by either interfering with protein synthesis or by inducing protein degradation. Protein synthesis can be blocked at various levels. At the genomic level, protein-coding genes can be disrupted using genome-editing technologies such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. These technologies are all based on nucleases that can be targeted to a specific DNA sequence and introduce a frame-shift mutation, a premature stop codon, or delete the coding sequence1. At the post-transcriptional level, messenger RNAs (mRNAs) can be targeted for degradation using RNA interference (RNAi). To this end, sequence-specific small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are introduced into cells. These become incorporated into RNA-induced silencing complexes (RISC), which bind to and degrade the target mRNA2. At the translational level, morpholino oligomers can be used to block the translation of mRNAs. These antisense oligonucleotides bind to the target mRNA and block the progression of the translation initiation complex from the 5’ cap to the start codon3. While these techniques have been proven to be highly useful for studying various genes in different model systems, a common major limitation is that they are not suitable to deplete already synthesized proteins. In both metabolically inactive and long-lived metabolically active cells, certain proteins – particularly those in essential cellular structures – can persist for years after translation4. These long-lived proteins cannot be depleted by blocking protein synthesis at the gene or mRNA level. Another limitation of methods that act at the gene or mRNA level is the long delay between the time of their application and actual protein depletion, which is typically in the range of days. These methods are therefore not well suitable to investigate short-lived biological processes. For example, many regulatory proteins have multiple functions during different stages of mitosis, which takes only about 1 hour in HeLa cells5. If protein degradation is delayed, it is difficult to determine if the phenotypes are a direct consequence of protein depletion or a secondary consequence of earlier 2 defects6. Delays may also allow cells to activate compensatory mechanisms, which may modify or even mask the phenotypes7. To overcome these challenges, several methods have been developed that act directly on the protein level, targeting the protein itself for degradation. Some of these methods are based on controlling protein stability, for instance by fusing the target protein to destabilizing domains that are controlled by ligands8,9. Others are based on recruiting SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases to the target protein, such as binding of an auxin-inducible degron to the F-box protein TIR1, and binding of GFP- tagged proteins to a GFP nanobody fused to the F-box domain of Slmb10, 11. Proteins can also be perturbed acutely by knock-sideways approaches, in which proteins are targeted away from their principle site of action, for instance by tethering them to mitochondria12. However, all of these assays require the endogenous protein to be first replaced by a modified variant. They are hence not suitable for studying protein functions in all cell types. For instance, application of these methods in non-dividing primary cells would often require the generation of transgenic animals. This is time- consuming and not feasible for many species. For tag-free degradation, ligand- and peptide-based techniques have been developed, but the number of proteins that can be targeted with these methods is very limited13,14. To achieve acute depletion of any endogenous protein without prior modification, we recently developed a post-translational protein depletion method, which we called ‘Trim-Away’15. Trim-Away relies on an E3 ubiquitin ligase called TRIM2116. TRIM21 is involved in the intracellular immune response: it binds to antibody-bound pathogens and proteopathic agents and targets them for degradation17-19. The precise mode of action of TRIM21 is not yet fully understood, but it involves binding of TRIM21 to the Fc-region of an antibody and subsequent autoubiquitination of TRIM21. In a Trim-Away experiment, the high affinity of TRIM21 to the Fc-region of an antibody is exploited to target endogenous proteins for degradation. An antibody against the target protein is introduced, TRIM21 binds to the antibody-bound target protein and triggers the proteasome-mediated degradation of the antibody-antigen complex together with TRIM2120,21. Trim-Away has enabled us to degrade a wide variety of proteins within minutes of application in different cell types15. For instance, we could acutely deplete Rec8 in unmodified mouse oocytes15, an experiment that required complex genetics in the past22. We have also been able to deplete the intracellular signalling molecule NLRP3 in human primary macrophages, which has not been possible with nucleic acid- based depletion techniques in the past15. Here, we describe (1) the design of Trim-Away experiments; (2) the selection and preparation of reagents for Trim-Away experiments; (3) the procedures for Trim-Away in mouse oocytes and pre-implantation embryos; (4) the procedures for Trim-Away in primary cells and cell lines; and propose (5) quality controls for Trim-Away experiments. We also highlight and give advice on critical steps in the procedure. Overview of the Procedure For a successful Trim-Away experiment, a specific antibody that targets an intracellular protein of interest must be delivered into cells that contain TRIM21. If the endogenous levels of TRIM21 are not sufficient for protein degradation, TRIM21 must also be introduced together with or before antibody delivery. In this Protocol, we outline different methods for introducing antibody and TRIM21 into cells and give advice on how to preselect specific antibodies for a successful Trim-Away experiment. A routine Trim-Away experiment involves the following three events (Fig. 1): 1. Ensuring sufficient TRIM21 levels in target cells of interest: TRIM21 is widely expressed in different cell types23. Depending on the expression level of the target protein, endogenous 3 TRIM21 levels may be sufficient for Trim-Away15. But as TRIM21 is continuously degraded during
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